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1983 62: 180-185
F Carr, WD Medina, S Dube and JR Bertino resistanceGenetic transformation of murine bone marrow cells to methotrexate
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Genetic Transformation of Murine Bone Marrow Cells to
180 Blood, Vol. 62, No. 1 (July), 1983: pp. 180-185
Methotrexate Resistance
By Frank Cam. William D. Medina, Shyam Dube, and Joseph R. Bertino
Genetic transformation of murine bone marrow stem cells
to methotrexate resistance was achieved using a modified
calcium phosphate-DNA coprecipitation procedure. Bone
marrow cells were transformed by DNA derived from
methotrexate-resistant mouse 3T6 cells. In vivo selection
of drug-resistant bone marrow cells resulted from thrice
weekly injections of methotrexate (MTX) for a period of
6-8 wk. Following selection. dihydrofolate reductase activ-
ity encoded by the donor DNA species was easily detect-
able in extracts of recipient mouse spleens. In addition.
A NOVEL IDEA for reducing the toxicity of van-
ous anticancer drugs to bone marrow cells is to
induce drug resistance in these cells. The experiments
of Cline and coworkers’ suggested that methotrexate
(MTX) resistant murine bone marrow cells could be
selected for in vivo following injection of cells treated
in vitro with DNA from a MTX-resistant mouse cell
line (3T6). MTX-resistant bone marrow cells were
routinely observed in different mice in these expeni-
ments, despite the expected low frequencies of trans-
formation to MTX resistance (usually less than lO�
with cell lines in tissue culture).2 The induction of
resistance to MTX in the majority of mice, together
with the presumably small numbers of pluripotent
stem cells injected, suggested that the efficiency of
transformation for these cells was higher than for most
established cell lines. The generality of transformation
of bone marrow cells to MTX resistance has been
further investigated, and an improved transformation
procedure has been developed.
C�ells
MATERIALS AND METHODS
Bone marrow cells were obtained from femurs of 6-wk-old female
CBA and CBA/H-T6T6 mice (Jackson Laboratory, Bar Harbor,
ME) by flushing the marrow cavity with ice-cold McCoy’s SA
medium supplemented with 10% fetal calf serum (FCS, GIBCO,
Grand Island, NY) and I U/mI hepanin. Donor mice were treated
with 4 mg/kg vinblastine 3 days prior to sacrifice to increase the
number of marrow hemopoietic stem cells.3 Chinese hamster ovary
(CHO) wild type and MTX-resistant A29 cells2 (obtained from Dr.
From the Departments of Pharmacology and Medicine. Yale
School ofMedicine, New Haven, CT.
Supported in part by Research Grant CA 28852; J.R.B. is an
American Cancer Society Professor.
Submitted July 27, /982; accepted January 19, /983.
Address reprint requests to Dr. J. R. Bertino, Yale University
School of Medicine, Department of Pharmacology. 333 Cedar
Street, New Haven, CT 06510.
© I 983 by Grune & Stratton, Inc.
0006-497//83/6/06-0026$Ol.00/0
selection of methotrexate-resistant cells was indicated by
the persistence of spleen colony-forming units (CFU-S) in
drug-treated animals. Also. changes in ratios of mixed
syngeneic bone marrow cells derived from CBA and CBA/
T6T6 mice resulted from initial treatment of either cell
type with 3T6 DNA. These results confirm and extend the
observations of Cline and coworkers that normal bone
marrow cells can be genetically transformed to methotrex-
ate resistance.
L. Siminovitch) were grown in Dulbecco’s modification of Eagle’s
medium (DMEM) containing 10% FCS and 3x normal nonessential
amino acids. A29 cells (Pro 3 MTXRIII2) possess an increased
activity of mutant form of CHO dihydrofolate reductase (DHFR)
activity, which is more resistant to MTX inhibition than the wild
type enzyme.4 Mouse embryo 3T6 fibroblast cells (obtained from
Dr. R. Kellems) weregrown in Dulbecco’s MEM with 10% FCS and
4 x l0�� M MTX. These cells contain high levels of mutant DHFR,
which has reduced affinity for MIX compared to wild type enzyme.�
L5178Y cells (single copy, MTX-sensitive DHFR) were grown in
Fischer’s medium containing 10% horse serum. Prior to preparation
of DNA or chromosomes, CHO, 3T6, and L5178Y cells were
propagated as suspensions in spinner flasks.
The cell line, 416B (obtained from Dr. E. Scolnick and Dr. M.
Moore), is a biopotential murine hemopoietic line derived from
cultures (C57BL/6 x DBA/2)F of bone marrow cells that were
infected with Friend leukemia virus.6 The 416B cells were grown in
Fischers medium supplemented with 20% horse serum. Human
lymphoblast CCRF-CEM cells (obtained from H. Lazerus) were
grown in RPMI 1640 medium containing 10’�f horse serum.
DNA and Chromosome Preparations
DNA was prepared by a modification of the method of Pellicer
and coworkers;’ this procedure involves extraction of aqueous cell
lysates three times with phenol and then three times with ether.
Purified DNA preparations were at least 69 Kb in size, as estimated
after electrophoresis in agarose gels. Chromosomes were isolated
from CHO and A29 cells by a modification of the method of Lewis
and coworkers.’ Following disruption of cells by homogenization,
chromosome preparations were layered onto a shelfof I M sucrose in
homogenization buffer and centrifuged at 100 g for 10 mm. The
sucrose-buffer interface layer was then removed and diluted before
applying to another I M sucrose shelf. The chromosomes were then
pelleted at I ,000 g for 20 mm, and finally washed and resuspended in
H EPES-buffered saline prior to transformation.
Genetic Transformation of Cells
Initial treatment ofcells with DNA or chromosome coprecipitates
with calcium phosphate was based on the modification of Wigler and
coworkers.’#{176} For bone marrow cell experiments, 2 x 106 cells/mI in
50 ml McCoy’s 5A medium containing 10% FCS were treated
directly in a TI 50 flask with 5 ml of calcium phosphate coprecipitate
with DNA (100 �.Lg) or chromosomes (1-2 cell equivalents). The
cells were incubated for 4 hr and then pelleted and resuspended in
fresh medium. This suspension also included particles of the micro-
precipitate, and in preliminary experiments, injection of this suspen-
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MTX-RESISTANT MURINE BONE MARROW CELLS 181
sion resulted in the rapid death of recipient mice. Therefore, the
concentrated cell suspensions were layered onto a shelf of 9.6%
(w/v) sodium metnizoate (Lymphoprep, Accurate Scientific and
Chemical Company, New York) and centrifuged at 500 g for 10
mm. Under these conditions, the microprecipitate pelleted, leaving
the bone marrow cells in a band at the metnizoate-medium interface.
The cells were collected, diluted to 6 x 10’ ml in fresh medium, and
0.3 ml were injected into X-irradiated (875 rad) recipient mice.
Early experiments utilizing the above protocol with calcium
phosphate coprecipitates with Cl-JO or A29 DNA were unsuccessful
(see Results). Modifications designed for cells propagated in suspen-
sion were tested using genetic transformation of CCRF-CEM cells
to MTX resistance as a model system. The optimal protocol involved
treating cells at about l0’/ml with an equal volume of calcium
phosphate microprecipitate containing DNA (20 zg/ml) or chromo-
somes (1 cell equivalent). After 15 mm, the cells were pelleted and
resuspended in 5 volumes of fresh medium (-. 106 cells/mI). Follow-
ing an overnight incubation, the cells were collected and separated
from the microprecipitates on a sodium metnizoate shelf, as
described above. These cells were then incubated in fresh medium at
2 x 105/ml for 30 hr. and MTX was added at I06 M for 72 hr.
Finally, the cells were diluted with 1.5 volumes of 0.2% agar in
RPMI + 20% horse serum and plated in culture tubes. Attempts to
genetically transform munine 4l6B cells involved an identical proto-
col, except that 4 16B cells were exposed to 3 x I 0 ‘ M MTX for 168
hr before plating in agar.
Successful adaption of the above modified protocol for genetic
transformation of bone marrow cells involved treating 108 cells in 6
ml of McCoy’s 5A medium containing 10% FCS with 6 ml of a
calcium phosphate coprecipitate with either 3T6 or L5178Y DNA.
After 15 mm, the cells were diluted into 60 ml fresh medium and
incubated for a further 4-S hr at 37#{176}C.Isolation of DNA-treated
cells and subsequent injection into recipient irradiated CBA mice
was as described above. In experiments involving mixing CBA and
CBA/T6T6 cells, 0.2 ml of LSI78Y DNA-treated cells was mixed
with 0.1 ml of 3T6-treated cells prior to injection. The MTX
treatment regimen was initiated 4 days after injection of donor
marrow cells into recipient mice. All injections were administered
intraperitoneally on a Monday/Wednesday/Friday schedule.
CFU-S Assays
The number of spleen colony-forming cells (CFU-S) in bone
marrow of experimental animals was determined by injecting 5 x
IO� or l0� cells into previously irradiated (900 rad) CBA mice.”
Spleen colonies were scored after I 0- 12 days.
DHFR Assays
DH FR activities of mouse spleens were assayed using crude
extracts prepared as follows. Spleens were minced in 50 mM Tnis,
pH 7.5, 2.5 mM MgCI2, 150 mM KCI, and 100 �ig/ml 3 mM
dithiothreitol (0.2 mI/spleen). The extract was sonicated on ice with
4 1 5-sec bursts, using a Branson sonicator at setting 5. The sonicate
was then centrifuged at I 5,000 rpm for 30 mm at 4#{176}Cin a Sorvall
refrigerated centrifuge, and the supernatant was collected and
assayed directly. DI-IFR activities were measured in a Guilford
spectrophotometer at 37#{176}C.The assay mix, in I ml, included 100
mM Tris, pH 7.5, 150 mM KCI, I 00 zg/ml NADPH, 20 �sM FH2,plus the extract and drug solution. The oxidation of NADPH was
followed at 340 nm in 4 different assays simultaneously. Spleenextracts from experimental animals were usually assayed in thepresence and absence of MTX (3 x 10’ M) in simultaneous
readings recorded on the same chart. DI-IFR activities were esti-
mated from the slopes of the 0D3,,, versus time plots. One unit of
enzyme activity is defined as a I nmole of NADPH oxidized in I
hr.
Hematocrit Measurements
Hematocnits were measured using blood drawn from the retroor-
bital sinuses of live mice using hepaninized capillary tubes.’2
Chromosome Analysis
Analysis of chromosomes from bone marrow cells of CBA and
CBA/H-T6T6 mice was based on the method of Ford.’3 Mice were
injected intrapenitoneally with 4 mg/kg colcemid 70-90 mm before
sacrifice. Bone marrow cells were flushed from the femurs of freshly
killed mice with McCoy’s SA medium, 15% FCS, and I U/mI
heparin. Cells were swollen in 75 mM KCI for 30-35 mm at 37#{176}C
and fixed in ice-cold methanol:glacial acetic acid (3:1) for I hr
(fixative changed 3 times). Chromosome spreads were prepared by
dropping the cell suspension onto microscopic slides from a height of
2-5 feet. Final chromosome spreads were stained with Trypsin-
Wright’s stain.’4 Spreads of 50-100 chromosomes were scored for
each analysis.
RESULTS
Establishment of Methotrexate Regimen
for CBA Mice
An appropriate maximally tolerated dose regimen
for MTX was first established using CBA mice. This
was done by monitoring hematocrits and CFU-S levels
in live mice as an indication of the hemopoietic sup-
pression induced by various doses of MIX. Mice were
initially X-irradiated with 875 rad and injected on the
following day with 2 x iO� bone marrow cells fromvinblastine-treated CBA donors. Injections were mi-tiated 4 days later, and hematocrits were monitored
after 3 and 6 wk. Control experiments indicated that
blood sampling (up to 0. 1 ml) from these mice, even on
a weekly basis, had no effect on hematocnits and
CFU-S values. Table 1 shows the change in hemato-
crits and CFU-S values with different dose schedules.
The schedule chosen for selection of transformed bone
marrow cells involved treatment with 2 mg/kg in the
first week following irradiation, 6 mg/kg in the second
week, and I 2 mg/kg in the third and subsequent weeks
(2/6/12 regimen). The 0.5/2/4 regimen was similar
to that employed by Cline and coworkers in their
experiments.’ However, in our hands, this regimen hadno detectable hematologic toxicity.
Failure to Transform Bone Marrow Cells to
Methotrexate Resistance Using A29 DNA
and Chromosomes
Initial attempts to genetically transform bone mar-
row cells to MTX resistance involved the use of
calcium phosphate coprecipitates with A29 DNA or
chromosomes. Chinese hamster ovary A29 cells have
been shown to overproduce a mutant species of DHFR
that is more resistant to MTX inhibition than the wild
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182 CARR ET AL.
Table 1 . Effect of Methotrexate Treatment Schedules on
Hematologic Parameters
Hematocrit HematocritDose StheduCe of MTX (mg/kg)
(Week 3) (Week 6) CFU-S/10’ Cells
Week 1 Week 2 Weeks 3-6 (%) (%) (Week 6)
0 0 0 49 51 20±3
0.5 2 4 51 50 29±2
1 4 8 51 46 4±2
2 6 12 48 27 1±1
3 8 16 39 ND’ ND
4 10 20 36 ND ND
Results are means of at least three independent measurements ( ± SD
for CFU-S measurements). Standard deviations for hematocrits were all
within 1 5% of the means. The dose schedules involved Monday/
Wednesday/Friday (p.m.) injections, and hematocnits and CFU-S levels
were assayed on Thursdays (p.m.). as described in Materials and
Methods.
‘ND. not determined due to death of mice.
type enzyme.4 DNA and chromosomes prepared from
A29 cells can transform mouse LTK cells to MTX
resistance with efficiency in excess of I0�6.8 We have
routinely used genetic transformation of LTK cells as
a positive control in our experiments. Despite success-
ful transformation of these cells to MTX resistance
using A29 DNA or chromosomes, we were initially
unable to find any evidence for transformed MTX-
resistant CBA bone marrow cells using similar proto-
cols (see Materials and Methods). This conclusion was
based on hematocrit and CFU-S measurements.
Transformation ofHuman Lymphoblast Cells in
Suspension Culture to Methotrexate Resistance
Following our failure to transform bone marrow
cells to MTX resistance with A29 DNA or chromo-
somes, we decided to examine in more detail the gene
transfer procedure. In particular, we wished to opti-
mize the genetic transformation of cells grown in
suspension culture, a procedure that we and others’5
have observed to be less efficient than transformation
of cells attached to a substrate. We observed that only
about 20% of a freshly isolated bone marrow cell
suspension adheres to the surface of tissue culture
flasks in the 4-hr period employed for exposure to
calcium phosphate-DNA coprecipitates. Most spleen
colony-forming cells are detachable in the suspension
during this periods. Attempts were made to genetically
transform human lymphoblast CCRF-CEM suspen-
sion cells to MTX resistance using A29 DNA or
chromosomes. The standard transformation protocol
used previously failed to yield MTX-resistant CCRF-
CEM. However, a modified protocol, which involved
increasing the cell number and the volume of calcium
phosphate microprecipitate by a factor of 5 (see Mate-
rials and Methods), led to the isolation, in 5 of 6 plates,
of colonies of MTX-resistant CCRF-CEM cells
induced by A29 chromosomes. The DHFR activity
associated with one of the resulting cell lines was then
characterized to test for expression of the mutant A29
DHFR species. The specific activity of DHFR in the
putatively transformed CCRF-CEM was found to be
elevated about 36-fold compared to wild type CCRF-
CEM cells (Table 2). This correlated with the
observed survival of the transformed cells at iO� M
and l0� M MTX, despite selection in 106 M MTX
(data not shown). Titration of equal amounts of
enzyme activity with different doses of MTX revealed
that DHFR from transformed CCRF-CEM cells was
more resistant to MTX inhibition than wild type
CCRF-CEM enzyme and was similar in resistance to
A29 DHFR (Fig. I). These results strongly suggest
that the MTX-resistant CCRF-CEM cells were genet-
ically transformed by donor A29 chromosomes using
the modified transformation protocol.
Failure to Transform a Biopotential Hemopoietic
Cell Line
Following the successful transformation of suspen-
sion-cultured CCRF-CEM cells, we attempted to
genetically transform the biopotential hemopoietic cell
line, 416B6 with A29 chromosomes. The cell line may
be closely related to murine hemopoietic stem cell by
virtue of a virally induced block in differentiation, and
therefore may provide a good in vitro model for
proliferating stem cells. Despite the low plating effi-
ciency (0.5%) for 4l6B cells in soft agar, we were able
to effectively screen 3.2 x I 0� cells (equivalent to 6.4 x
l0� cells plated) for genetic transformation to MTX
resistance by A29 chromosomes. However, no colonies
were isolated after exposure to 3 x iO� M MTX,
suggesting a transformation efficiency with A29 chro-
mosomes of less than 3 x 106. Other experiments
aimed at genetically transforming freshly isolated
bone marrow cells to MTX resistance, as judged by the
appea rance of MTX -resista nt gra nulocyte- macro-
phage colonies (CFU-C) in methylcellulose’6 (plating
efficiency of 0. 1%), were also unsuccessful, having
effectively screened 8 x I O� cells.
Table 2. Comparative DHFR Activities of Hamster and Human
Cell Lines
Cell Line
DHFR Activity
(U/mg Protein)
CHO 98
A29 (CHO MTX”) 4,832
CCRF-CEM 80
CCRF-CEM MTX 2.9 12
Enzyme activities are expressed in units. One unit is defined as 1 nmole
of NADPH oxidized per hour. The activities are means for 3 estimations,
with standard deviations of less than 20%.
For personal use only. by guest on December 5, 2013. bloodjournal.hematologylibrary.orgFrom
00
>‘
.� 80
C.)
a: 60LLI0
20
[MTx], M
100 �
�‘
:� \3T6- .) mouse \c-i
< spleen #{149}\a: 50U-
a�2 25
0 � i
IO� l0�� lO� 106 lO� �
[ Mlxi, M
Fig. 2. Titration of mouse spleen DHFR and 3T6 DHFR byMTX. DHFR assays were run in the absence or presence ofincreasing amounts of MTX. as described in Materials and Meth-ods.
MTX-RESISTANT MURINE BONE MARROW CELLS 183
Fig. 1 . Titration of DHFR activity for human (CEM/CEM MIX) and hamster (CHO/A29) cell lines withMTX. Enzyme activities were measured at least in
triplicate (mean shown) by monitoring the oxidation ofNADPH at 340 nm. Approximately 5 U of enzyme wereassayed for each cell line. One unit is defined as theamount of enzyme required to reduce 1 nmole ofdihydrofolate in 1 hr. (0) CEM; (�) CHO; (S) A29; (0)CEM MTX; cells transformed with A29 chromosomes.
40
Transformation ofBone Marrow Cells to
Methotrexate Resistance With 3 T6 or pHG DNA
The choice of donor A29 genetic material for pre-
vious bone marrow transformation experiments was
based on the observation that mutant A29 DHFR
genes can efficiently induce easily selectable trans-
formed MTX-resistant mouse cells.8 The previously
successful genetic transformation of mouse bone mar-
row cells by Cline and coworkers’ involved treatment
of marrow cells with DNA from 3T6 cells. Recent
studies have revealed that 3T6 cells produce a mutant
DHFR activity over 100-fold resistant to MTX inhibi-
tion compared to the wild type enzyme activity.5 This
represented a proportionally greater MTX resistance
than was found for the DHFR activities of A29 cells
compared to wild type CHO cells (15-fold; Fig. I).
Studies with the 3T6 subline, induced in this labora-
tory to grow in 2 mM MTX, revealed an even greater
relative resistance ( I 0,000-fold) of 3T6 DH FR activi-
ties to MTX (Fig. 2). The mutant enzyme activity in
this case titrated over a relatively narrow dose range of
MTX (l0��-l0 “ M) compared with the previously
characterized 3T6 activity ( l0��-l0� M).3
The presence ofone or more mutant DHFR genes in
3T6 cells might explain the success of the bone marrow
cell transformation experiments of Cline and cowork-
ers.’ Therefore, we subsequently used 3T6 DNA in
attempts to transform bone marrow cells to MTX
resistance. For these experiments, we utilized mixtures
ofsyngeneic CBA and CBA/T6T6 bone marrow cells,’
which are easily distinguishable by chromosomal anal-
ysis.’3 Mixtures of cells treated with calcium phos-
phate coprecipitates with 3T6 DNA and L5178Y
DNA in ratios of 1:2 were injected into irradiated
recipient CBA mice (Table 3). Hematocnits were
measured 43 days after injection, and the mice were
sacrified between days 46 and 58. About half of the
mice were treated with colcemid 70-90 mm before
sacrifice, and bone marrow cells from these mice were
used for chromosomal analysis. Bone marrow cells
from the remaining mice were used for CFU-S assays.
The spleens were removed from every experimental
mouse and utilized for DHFR assays. Enzyme assays
for DI-IFR were performed in the presence or absence
of 3 x 10 � M MTX for animals injected with 3T6
DNA-treated marrow cells. This dose is completely
inhibitory to wild type mouse DHFR enzyme, while it
does not inhibit the activity from 3T6 cells (Fig. 2).
A summary of results of all of the analyses per-
formed on mice injected with 3T6-treated bone mar-
row cells is provided in Table 3. In every case, animals
transformed with 3T6 DNA displayed elevated hema-
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184 CARR ET AL.
Table 3. Analysis of CBA Mice Injected With Calcium Phosphate-DNA Treated Bone Marrow Cells and Treated With MTX
for 46-58 Days
Final Percent Final
(A) DNA
Treatment
)CBA)Percent
Hematocrit
Percent 3T6
DHFR
Activity
Percent
CBA
(Marrow)
CFU-S
(per 10’ Cells)
I ± SD)
(B) DNA
Treatment
)CBA/H-T6T6)Percent
Hematocrit
3T6
DHFR
Activity
Percent
CBA
(Marrow)
CFU-S
(per iO’ Cells)
( ± SD)
None 45-53 0 (33) 30(±4) None 46-53 0 (33) 30(±4)
3T6 3T6
Mouse no. 1 34.5 39 60 Mouse no. 6 30 18 48
2 35.5 33 48 7 39.5 28 40
3
4
5
50
39.5
39
78
45
36
93
30(±17)
14(±3)
8
9
10
39
32.5
35
26
0
14
13
0
0
L5178Y 24
29
29.5
0
0
4
33
10
1(±2)
11
L5178Y
36
23.5
26
28
0
4
0
18(±9)
24
34
25
27
31
26
29.5
0 32
0
Hematocrits were measured after 43 days with blood drawn from the retroorbital sinus of donor mice. DHFR activities, measured as rates of change of
340 nm absorption, are expressed as percentage activity ratios in the presence or absence of 3 x 10 � � M MTX (from one analysis only). Activity of DHFR
in the absence of MTX was considered to be 1 00% . The ratio of CBA or CBA/H-T6T6 bone marrow cells was calculated from at least 1 00 chromosome
spreads for each analysis. This ratio is presented in the table as the percent of CBA chromosomes found of the total. CFU-S levels for each individual
mouse are expressed as means for 3-6 separate analyses.
tocrits compared to control animals injected with
untreated or L5178Y DNA-treated cells only. This
increase was striking for animal 3T6-3, which dis-
played a hematocrit in the range of uninjected con-
trols. In spleens derived from all but one animal
(3T6-9), DHFR activities characteristic of those
encoded by 3T6 genes were detected, indicating that
these genes were being expressed in the spleens of
positive animals. Chromosomal analysis of bone mar-
row cells revealed a significantly increased relative
proliferation of 3T6 DNA-treated marrow cells com-
pared to L5178Y-treated cells in all animals except
3T6-8. Finally, spleen colony-forming units derived
from marrow cells of the 3T6 series mice were detected
in all cases, except from mice 3T6-9 and 3T6-l0.
Therefore, at least two positive results suggesting
the presence of transformed MTX-resistant bone mar-
row cells were obtained for all animals, apart from
mice 3T6-9 and 3T6- I 0. These mice displayed slightly
elevated hematocrits, for reasons that are presently
unclear. The results indicate that treatment of mouse
bone marrow cells with calcium phosphate coprecipi-
tates and 3T6 DNA, using a modified protocol, gives
rise to transformed MTX-resistant cells at high effi-
ciency.
DISCUSSION
These results confirm the observations of Cline and
coworkers’ that MTX-resistant mouse bone marrow
cells can be efficiently produced in vivo by transforma-
tion with calcium phosphate-3T6 DNA coprecipitates
in vitro. Several improvements in the protocol used by
Cline and coworkers have been implemented includ-
ing: (A) exposure of more concentrated cell suspen-
sions to increased volumes ofcalcium phosphate-DNA
coprecipitates; (B) separation of these coprecipitates
from bone marrow cells prior to injection into recipient
mice; and (C) increasing the doses of MTX used for in
vivo selection of MTX-resistant transformed bone
marrow cells.
These improvements were shown to increase the
efficiencies of the genetic transformation, bone mar-
row cell administration, and bone marrow cell selection
steps in the protocol. As a result, genetically trans-
formed MTX-resistant bone marrow cells were
detected in 9/ 1 1 mice that had received 3T6-treated
marrow cells (Table 3). However, no genetically trans-
formed cells were detected in mice injected with A29
DNA-treated marrow cells or with L5178Y DNA.
The high level of resistance of the mutant DHFR
activity encoded by 3T6 DNA may have permitted
proliferation of 3T6 DNA-transformed cells in vivo in
conditions where A29 DNA-transformed cells were
unable to proliferate. In addition, in view of the
markedly altered 3T6 R400 DI-IFR gene, it is likely
that transfection with only a single 3T6 gene would
impart MTX resistance to the recipient marrow stem
cell.
We are currently attempting to isolate the cDNA
for DHFR from 3T6 cells for future gene transferexperiments. It should be possible, using the transfor-
mation procedure developed, to transfer almost any
cloned gene to mouse bone marrow cells without
impairing the proliferation capacity of these cells in
vivo. Genes coding for nonselectable markers may
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MTX-RESISTANT MURINE BONE MARROW CELLS 185
potentially be transferred to bone marrow cells with
selectable genes,”8 such as those encoding DHFR and
thymidine kinase.’9 Alternative gene transfer proce-
dures, such as cell-to-cell fusion and liposome-
mediated gene transfer, are more likely to impair the
normal functioning of these cells.
The transfer of drug-resistant genes to normal bone
marrow cells perm its i ncreased hematologic tolerance
to intensive drug treatment schedules, as demonstrated
in these studies. This tolerance may improve thera-
peutic ratios for treatment of a variety of tumors with
common anticancer agents, and experiments are
planned to assess this possibility using model tumors in
mice. A major drawback of the present method for
induction of MTX resistance in bone marrow cells is
that a relatively long selection time is necessary to
amplify MTX-resistant cells to detectable levels. Fur-
then improvements in transformation efficiencies are
therefore desirable. However, it is likely that an appre-
ciable number of bone marrow cells initially express
donor DHFR genes shortly after gene transfer, as
demonstrated in other systems.2#{176} This initial transient
expression may explain the elevated hematocrits
observed for mice 3T6-9 and 3T6- I 0 (see Table 3) in
the absence of detectable donor DHFR activity in the
spleen. Therefore, intensive chemotherapy may be
possible immediately after receiving bone marrow cells
transformed with genes concerning drug resistance.
This idea is currently being investigated.
The high efficiency of bone marrow cell transforma-
tion to MTX resistance observed in the experiments
reported supports the conclusion of Cline and cowork-
ers’ that normal munine bone marrow is as transform-
able as some tissue-cultured cell lines. Therefore, the
mouse system provides a good model system with
which to study the potential clinical applications of
bone marrow cell transformation. Further characteri-
zation of this genetic transformation system is neces-
sary prior to adaption of these procedures to humans.
The location of the 3T6 R400 DNA in the marrow
cells has not yet been determined. One explanation for
the transient resistance of mice 9 and I 0 to MTX could
be that the 3T6 DNA was extrachromosomal and not
integrated into host chromosomes.
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